BCL-2 family proteins are critical regulators of apoptosis, a form of programmed cell death that is essential to tissue development, homeostasis, and organism longevity. MCL-1 is an anti-apoptotic BCL-2 protein that binds and inhibits select pro-apoptotic members. Loss of MCL-1 function can sensitize cells to apoptosis and lead to premature cell death, whereas over-expression can prolong cell survival in the face of pro-apoptotic stress. MCL-1 is unique among the anti-apoptotics both in its interaction profile and complex post-translational regulation. Whereas small molecules have been developed to target the BCL-2 subclass of anti-apoptotic proteins, selective modulators of MCL-1 have remained out of reach. Because such compounds would be valuable tools to analyze MCL-1 function in health and disease, we undertook a screening assay and identified a class of compounds that bound to MCL-1 selectively and covalently, leading to striking changes in MCL-1 structure and function. In characterizing the binding sites, cysteine modification emerged as a chemical mechanism for MCL-1 modulation with potential physiologic implications. Here, I propose to examine the biochemical and cellular consequences of cysteine modification on MCL-1 activity, which could reveal a new druggable mechanism for maintaining cell survival in the face of premature or unwanted cell death. I will take a multidisciplinary approach to explore my hypothesis that cysteine modification can regulate the anti-apoptotic activity of MCL-1. To define the potential sites of modification, I will examine the chemical reactivity of individual cysteine residues in recombinant MCL-1. I will then determine the effect of cysteine modification on the functional activity of MCL-1 by testing its BH3-binding activity and capacity to block BAK- mediated mitochondrial apoptosis. I will define the mechanism underlying the functional changes by determining the x-ray structure of cysteine-modified MCL-1. To extend the investigation to a cellular context, I will examine the cysteine-derivatization state of native MCL-1 and probe the in situ consequences of cysteine modification by reconstituting MCL-1-/- cells with MCL-1 and cysteine mutants. I will screen for changes in MCL-1 activity in response to stress stimuli, monitoring cellular viability and biochemical markers of apoptosis. Given the seminal role of MCL-1 in cell survival, I am eager to probe a potentially fundamental mechanism of MCL-1 regulation and uncover a novel strategy to influence cellular longevity for therapeutic benefit. I look forward to acquiring new multidisciplinary skills at the interface of chemical biology, apoptosis biology, and aging research. The proposed training plan at Dana-Farber and Harvard Medical School offers state-of-the-art resources, world class faculty advisors and collaborators, and an outstanding environment to facilitate my continued scientific growth. I am committed to a scientific career focused on the structure and function of protein interactions that regulate apoptosis, with direct application to the development of novel pharmacologic strategies to protect cells from pathologic cell death. PUBLIC HEALTH RELEVANCE: BCL-2 family proteins regulate programmed cell death, or apoptosis, and function as a molecular rheostat that controls cell survival during the aging process. MCL-1 is a powerful pro-survival BCL-2 family protein that is subject to exquisite cellular control, and when deregulated can cause premature cell death or pathologic cell survival. In this proposal, I will use unique chemical tools, structure-function analyses, and cell survival studies to investigate a new mechanism for regulating MCL-1, with the goal of advancing a novel pharmacologic strategy for cytoprotection to combat age-related cellular stress.